Real time binding analysis of antigens on a biosensor surface

ABSTRACT

The invention provides methods for detecting interactions between phage and antigen or antigen and antibody using biosensors.

PRIORITY

This application is a divisional application of U.S. Ser. No.11/290,036, filed Nov. 30, 2005, (now allowed), which claims priority toU.S. Ser. No. 10/399,940, filed Jan. 16, 2004, now U.S. Pat. No.7,202,076, which is a continuation of PCT/US01/45455, filed Oct. 23,2001, which is a continuation in part of U.S. Ser. No. 09/930,352, filedAug. 15, 2001, now U.S. Pat. No. 7,094,595, which claims the benefit ofU.S. Ser. No. 60/303,028 filed Jul. 3, 2001; U.S. Ser. No. 60/283,314,filed Apr. 12, 2001; and U.S. Ser. No. 60/244,312, filed Oct. 30, 2000.U.S. Ser. No. 11/290,036, filed Nov. 30, 2005, also claims priority toPCT/US03/01298, filed Jan. 16, 2003, which is a continuation of U.S.Ser. No. 10/059,060, filed Jan. 28, 2002, now U.S. Pat. No. 7,070,987,which is a continuation in part of U.S. Ser. No. 09/930,352, filed Aug.15, 2001, now U.S. Pat. No. 7,094,595. U.S. Ser. No. 11/290,036, filedNov. 30, 2005, also claims priority to PCT/US03/01298, filed Jan. 16,2003, which is a continuation in part of U.S. Ser. No. 10/058,626, filedJan. 28, 2002, which is continuation in part of U.S. Ser. No.09/930,352, filed Aug. 15, 2001, now U.S. Pat. No. 7,094,595, all ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of biosensors and methods comprisingdetecting antigens that specifically bind to an antibody, antibodyfragment, or phage.

BACKGROUND OF THE INVENTION

The ability to detect binding between phage and mammalian cells is anessential component for discovery of therapeutic and diagnosticantibodies. A typical pipeline for identifying potential therapeutic anddiagnostic antibodies includes: (1) phage display and phage panningexperiments on soluble protein or cellular associated proteins (in thesoluble form or expressed on cells); (2) a phage ELISA performed onsoluble protein (for cellular targets—a peptide or protein-mimic of thecellular associated protein); (3) the display gene in the phage genomeis subcloned via molecular biology techniques to a soluble antibodyfragment expressing plasmid; (4) The antibody fragment then is expressedand purified; (5) once purified the antibody fragment can be tested forcellular functional binding in ELISA, FACS, Guava or FMAT; (6) The leadantibody fragment is analyzed for binding kinetics; and (7) the topantibody lead is then cloned into a full antibody expression vector forlarge scale production, kinetic analysis and in vivo efficacy models.

Typical assays for analysis of functional binding of phage to proteintargets associated with cells include whole mammalian or bacterial cellenzyme-linked immunosorbent assay (ELISA), flow cytometry (FluorescenceActivated Cell Sorter, FACS), Guava microcytometry products (GuavaTechnologies, Hayward, Calif.), and fluorescence microassay technology(FMAT). ELISAs have high background binding of phage, because cells arecomplex and phages have a tendency to bind non-specifically. Backgroundbinding in ELISA is intensified due to amplification of the bindingsignal. Cellular ELISAs are also difficult due to the need of manywashes between steps, which is cumbersome if the cells are non-adherentas a centrifugal spin is required between each wash. Often adherentcells must be fixed in order to keep the cells attached to the ELISAplate during washes, either manually or on a plate washer. The fixationcan change the natural epitopes of the protein on cells. Phage bindingin FACs and Guava is also difficult, because each phage clone needs tobe purified to get enough phage for a signal.

Currently, most researchers spend a lot of time and effort in cloningthe display on the phage to fragments and/or full IgGs in order toinvestigate the functional binding to cells. The more time spentidentifying potential therapeutic antibodies, the longer it takes to geteffective therapeutic antibodies into medical clinics. Thus, there is aneed in the phage display field for a quick route to identifyingfunctional binding of antibodies to mammalian cells.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a method of detecting bindingof a binding partner to a phage. The method comprises immobilizing acrude phage preparation, unconcentrated phage preparation,non-homogenous phage preparation, or a combination thereof on abiosensor and contacting the biosensor with the binding partner. Bindingof the binding partner to a phage immobilized on the biosensor isdetected. The binding partner can be a small molecule, a carbohydrate, apolymer, a peptide, a soluble protein, a cellular receptor, an antigenmimic of a cellular receptor, a cell, a mammalian cell, or a mammaliancell surface protein. The phage preparation and antigen do notnecessarily comprise a detectable label. The phage preparation can be aphage display library. The phage preparation can be passivelyimmobilized to the biosensor or can be immobilized to the biosensor byan antibody specific for a phage coat protein. The antibody or antibodyfragment can be immobilized to the biosensor by binding to a proteinthat is bound to the biosensor. If the antibody or antibody fragmentcomprises a tag, the antibody or antibody fragment can be immobilized tothe biosensor by antibodies specific for the tag. The biosensor can be acalorimetric resonant reflectance biosensor or an evanescent wave-basedbiosensor.

Another embodiment of the invention provides a method for determiningepitope classes of antibodies in an antibody population. The methodcomprises immobilizing a display phage, antibody, or antibody fragmentto a biosensor and contacting the biosensor with a binding partner thatspecifically binds to the display phage, antibody, or antibody fragmentimmobilized to the biosensor, under conditions suitable for binding ofthe binding partner to the display phage, antibody, or antibodyfragment. The antibody population is contacted with the biosensor. Adetectable signal generated by binding of the antibody population to thebinding partner indicates that different epitope classes are present inthe antibody population than in the immobilized display phage, antibody,or antibody fragment. The antibody population, binding partner andimmobilized display phage, immobilized antibody, or immobilized antibodyfragment do not necessary comprise a detectable label. The antibodypopulation can comprise phage clones, antibody fragments, fullantibodies, phage displaying a full antibody, phage displaying anantibody fragment, antibodies from a hybridoma, and antibodies from aphage display screen. The display phage can be a purified phagepreparation, a crude phage preparation, an unconcentrated phagepreparation, or a non-homologous phage preparation. The binding partnercan be a small molecule, a carbohydrate, a polymer, a peptide, a solubleprotein, a cellular receptor, an antigen mimic of a cellular receptor, amammalian cell, or a mammalian cell surface protein. The mammalian cellsurface protein can be a membrane-associated protein, a singletransmembrane protein, a multi-transmembrane protein, or a proteinchannel. The biosensor can be a calorimetric resonant reflectancebiosensor or an evanescent wave-based biosensor.

Therefore, the invention provides methods to, e.g., resolve lowconcentrations of binding partners, rank protein affinities, work withsamples comprising complex mixtures, and perform off-rate rankinganalysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C depicts an antibody and antibody fragments, F(ab) and scFv.FIG. 1A shows a full IgG antibody and domains of the IgG. FIG. 1B showsF(ab). FIG. 1C shows scFv.

FIG. 2 shows titration of bacterial viruses on GA3 BIND® Biosensor

FIG. 3A-B shows F(ab) capture from periplasmic extract on a TIO BIND®Biosensor. FIG. 3A shows creation of the sF(ab) specific capture surfaceand the capture of sF(ab). FIG. 3B shows a graphical representation ofthe capture of sFab recorded in FIG. 3A.

FIG. 4A-D shows scFv capture from periplasmic extract on a TIO BIND®Biosensor. FIG. 5A shows the creation of scFv specific capture surfaceand capture of scFv containing a 6×His and c-myc tag. FIG. 4B showscapture of purified scFv spiked into PBS and periplasmic extract. FIG.4C shows a graphical representation of capture of scFv recorded in FIG.4B. FIG. 4D shows graphical representation of capture of scFv recordedin FIG. 4B.

FIG. 5A-C shows scFv capture from periplasmic extract on a SA1 BIND®Biosensor. FIG. 5A shows creation of the specific capture surface forproteins expressing a 6×his tag. FIG. 5B shows capture of scFv spikedinto PBS and periplasmic culture. FIG. 5C shows graphical representationof scFv capture of purified scFv spiked into PBS and periplasmicextract.

FIG. 6A-C shows scFv capture from periplasmic extract on a GA1 BIND®Biosensor. FIG. 6A shows creation of specific capture surface forproteins containing a c-myc tag. FIG. 6B shows capture of purified scFvspiked into PBS and a periplasmic extract. FIG. 6C shows a graphicalrepresentation of scFv capture from PBS and periplasmic extract.

FIG. 7A-E shows capture of IgGS from hybridoma supernatants using ananti-Fc TIO BIND® Biosensor. FIG. 7A shows creation of specific mouseIgG capture surface. FIG. 7B shows creation of specific mouse IgGcapture surface. FIG. 7C shows capture of IgGs from hybridoma media.FIG. 7D shows a graphical representation of antigen, antigen and cells,and cells binding to the IgGs captured on the biosensor surface. FIG. 7Eshows a tabular representation of antigen, antigen and cells, and cellsbinding to the IgGs captured on the biosensor surface with thenormalization of this binding to the amount of IgG captured on thebiosensor surface.

FIG. 8A-C shows IgG capture from serum using an anti-Fc TIO BIND®Biosensor. FIG. 8A shows creation of a specific mouse IgG bindingsurface. FIG. 8B shows capture of IgGs from serum. FIG. 8C shows captureof IgGs from serum.

FIG. 9A-C shows a drug—anti-drug assay in serum using a GA1 BIND®Biosensor. FIG. 9A shows creation of a surface for the capture of anIgG, an anti-drug. FIG. 9B shows capture of the anti-drug from PBS, 11%and 30% serum on the 20 ug/ml drug surface. FIG. 9C shows capture of theanti-drug from PBS, 11% and 30% serum on the 0 ug/ml drug surface.

FIG. 10A-C shows IgG capture from serum using an anti-Fc TIO BIND®Biosensor. FIG. 10A shows creation of the drug surface for the captureof anti-drug. FIG. 10B shows capture of the anti-drug from PBS, 11% and30% serum on the 50 ug/ml Drug surface. FIG. 10C shows capture of theanti-drug from PBS, 11% and 30% serum on the 0 ug/ml drug surface.

FIG. 11A-D shows endpoint analysis of antibody binning andidentification of sandwich pairs of antibodies on an anti-FC TIO BIND®Biosensor. FIG. 11A shows creation of mouse IgG specific surface. FIG.11B shows capture of layer 1 (Antibody). FIG. 11C shows capture of layer2 (Antigen by Antibody). FIG. 11D shows capture of layer 3 (Antibody byAntibody-Antigen Complex).

DETAILED DESCRIPTION OF THE INVENTION

Biosensors

In one embodiment, the methods of the invention comprise the use of abiosensor that can be used to, inter alia, detect inorganic or organicmaterial, such as protein, DNA, small molecules, viruses, cells, andbacteria, without the requirement of a detectable label, such asfluorescent or radioactive labels. Numerous suitable biosensors can beused in the methods of invention, including, but not limited to,photonic crystal biosensors (e.g., colorimetric resonant reflectancebiosensors, silver nanoparticle array biosensors), interferometricbiosensors (e.g., RIfS, dual polarization interferometer, HartmanInterferometer), MEMS biosensors (e.g., cantilevers, resonantmembranes), acoustic biosensors (e.g., quartz resonator), microwavebiosensors (e.g., dielectric spectroscopy), surface plasmon resonance(SPR) biosensors (e.g., kreitchman SPR biosensors, imaging SPRbiosensors, grating coupled imaging SPR biosensors), waveguidebiosensors (e.g., input grating coupler biosensors, chirped waveguidegrating biosensors), evanescent wave-based biosensors and any biosensorsincorporating an optical waveguide, as described for example in U.S.Pat. Nos. 4,815,843; 5,071,248; and 5,738,825; the disclosures of whichare incorporated by reference in their entirety.

The methods of the invention have utility in, inter alia, the fields ofpharmaceutical research (e.g., primary screening, high throughputscreening, secondary screening, quality control, cytotoxicity, clinicaltrial evaluation), life science research (e.g., proteomics, proteininteraction analysis, DNA-protein interaction analysis, enzyme-substrateinteraction analysis, cell-protein interaction analysis), diagnostictests (e.g., protein presence, cell identification), and environmentaldetection (bacterial and spore detection and identification).

Previous patent applications and publications describe how acolorimetric resonant reflectance biosensor surface, in combination witha high resolution imaging instrument, can be used as a platform forperforming many biochemical assays in parallel upon on single surface,using only nanoliters of sample material. See, e.g., U.S. Pat. Publ.Nos.: 2002/0168295; 2002/0127565; 2004/0132172; 2004/0151626;2003/0027328; 2003/0027327; 2003/017581; 2003/0068657; 2003/0059855;2003/0113766; 2003/0092075; 2003/0026891; 2003/0026891; 2003/0032039;2003/0017580; 2003/0077660; 2004/0132214.

Colorimetric resonant reflectance biosensors comprise a subwavelengthstructured surface. Subwavelength structured surfaces are a type ofdiffractive optic that can mimic the effect of thin-film coatings. See,e.g., Peng & Morris, “Resonant scattering from two-dimensionalgratings,” J. Opt. Soc. Am. A, Vol. 13, No. 5, p. 993, May 1996;Magnusson, & Wang, “New principle for optical filters,” Appl. Phys.Lett., 61, No. 9, p. 1022, August, 1992; Peng & Morris, “Experimentaldemonstration of resonant anomalies in diffraction from two-dimensionalgratings,” Optics Letters, Vol. 21, No. 8, p. 549, April, 1996. Agrating of a photonic crystal biosensor of the invention has a gratingperiod that is small compared to the wavelength of incident light suchthat no diffractive orders other than the reflected and transmittedzeroth orders are allowed. A photonic crystal biosensor can comprise agrating, which is comprised of or coated with a high dielectric constantdielectric material, sandwiched between a substrate layer and a coverlayer that fills the grating grooves. Optionally, a cover layer is notused. The grating structure selectively couples light at a narrow bandof wavelengths. This highly sensitive coupling condition can produce aresonant grating effect on the reflected radiation spectrum, resultingin a narrow band of reflected or transmitted wavelengths. The depth andperiod of the grating are less than the wavelength of the resonantgrating effect.

The reflected or transmitted color of a calorimetric resonantreflectance biosensors structure can be modified by the addition ofmolecules. The added molecules increase the optical path length ofincident radiation through the biosensor structure, and thus modify thewavelength at which maximum reflectance or transmittance will occur.

When illuminated with white light a calorimetric resonant reflectancebiosensor reflects only a single wavelength or a narrow band ofwavelengths. When molecules are attached to the surface of thebiosensor, the reflected wavelength (color) is shifted due to the changeof the optical path of light that is coupled into the grating. Byimmobilizing molecules, such as specific binding substances to abiosensor surface, complementary binding partner molecules can bedetected without the use of any kind of detectable label, e.g., afluorescent probe or particle label. The detection technique can beperformed with the biosensor surface either immersed in fluid or dried.

When a colorimetric resonant reflectance biosensor is illuminated withcollimated white light and reflects only a narrow band of wavelengths,or a single band of wavelengths is reflected. The narrow wavelength bandis described as a wavelength “peak.” The “peak wavelength value” (PWV)changes when molecules are deposited or removed from the biosensorsurface. A readout instrument illuminates distinct locations on thebiosensor surface with collimated white light, and collects collimatedreflected light. The collected light is gathered into a wavelengthspectrometer for determination of PWV.

Evanescent wave-based biosensors can comprise a waveguiding filmsupported by a substrate; between the waveguiding film (and optionallyas part of the substrate) is a diffraction grating. See, e.g., U.S. Pat.No. 4,815,843. A low-k dielectric material, such as low-k nanoporousmaterial can be used for the diffraction grating or the combined low-knanoporous material and substrate. The waveguide comprises waveguidingfilm and the substrate. The waveguiding film can be, e.g., tin oxide,tantalum pentoxide, zinc sulfide, titanium dioxide, silicon nitride, ora combination thereof, or a polymer such as polystryrole orpolycarbonate. A diffraction grating exists at the interface of thewaveguiding film and the substrate or in the volume of the waveguidingfilm. The diffraction grating comprises a low-k material, such as low-knanoporous material. The refractive index of the waveguiding film ishigher than the index of the adjacent media (i.e., the substrate and thetest sample). The substrate can be, e.g., plastic, glass or epoxy. Aspecific binding substance can be immobilized on the surface of thewaveguiding film and a test sample added to the surface. Laser lightpropagates in the waveguiding film by total internal reflection. Changesin refractive index of the waveguiding film caused by molecules bindingto it can be detected by observing changes in the angle of the emitted,out-coupled light.

A biosensor of the invention can comprise an inner surface, for example,a bottom surface of a liquid-containing vessel. A liquid-containingvessel can be, for example, a microtiter plate well, a test tube, apetri dish, or a microfluidic channel. One embodiment of this inventionis a biosensor that is incorporated into any type of microtiter plate.For example, a biosensor can be incorporated into the bottom surface ofa microtiter plate by assembling the walls of the reaction vessels overthe resonant reflection surface, so that each reaction “spot” can beexposed to a distinct test sample. Therefore, each individual microtiterplate well can act as a separate reaction vessel. Separate chemicalreactions can, therefore, occur within adjacent wells withoutintermixing reaction fluids, and chemically distinct test solutions canbe applied to individual wells.

The most common assay formats for pharmaceutical high-throughputscreening laboratories, molecular biology research laboratories, anddiagnostic assay laboratories are microtiter plates. The plates arestandard-sized plastic cartridges that can contain 96, 384, or 1536individual reaction vessels arranged in a grid. Due to the standardmechanical configuration of these plates, liquid dispensing, roboticplate handling, and detection systems are designed to work with thiscommon format. A biosensor of the invention can be incorporated into thebottom surface of a standard microtiter plate. Because the biosensorsurface can be fabricated in large areas, and because the readout systemdoes not make physical contact with the biosensor surface, an arbitrarynumber of individual biosensor areas can be defined that are onlylimited by the focus resolution of the illumination optics and the x-ystage that scans the illumination/detection probe across the biosensorsurface.

Phage Preparations

Phage are bacterial viruses and are species specific. For thisparticular discussion, two Escherichia coli phage, M13 and Lambda, arebeing discussed. In the case of other bacterial phage and/or mammalianviruses the same premise applies. A population of phage, in particular,a non-homogenous, crude, and/or unconcentrated population of phage, suchas a phage display library can be used in methods of the invention. Anon-homogenous preparation of phage comprises a preparation thatcontains one or more type of phage, e.g., a phage display librarywherein each phage displays a different binding molecule. A crude phagepreparation is a preparation that contains one or more types of phage inthe medium in which bacteria infected with phage was grown in. In thecase of M13, the phage fuse through the membrane and the cell is notlysed. In the case of lambda, the cells are lysed and the medium wouldcontain cellular components. In this case the crude phage preparationwould be clarified of bacterial cells and membrane components bycentrifugation. A crude phage preparation contains one or more types ofphage at low concentrations in the presence of media components andexcreted cellular catabolites.

An unconcentrated phage preparation is a phage preparation where thephage has not been precipitated. When the phage is precipitated, themedium is removed and the phage can be resuspended in a defined buffersuch as PBS. During the purification process of M13 phage, the phage isusually precipitated with a PEG solution one or two times and is storedin PBS glycerol. During the process the phage is resuspended in smallervolumes of buffer than the original volume of medium. A typical 1-2liter culture of medium will be resuspended in a final volume of about2-5 ml of PBS, providing a purified and concentrated stock of phage.

Immobilization of Phage

A phage preparation is immobilized to a biosensor. A phage preparationcan be passively immobilized to a biosensor surface. The phage surfacecan be blocked and an antigen (e.g. small molecule, carbohydrate,polymer, peptide, soluble protein, antigen mimic of a cellular receptor,or mammalian cells) can be screened for binding to the immobilizedphage. The binding of the antigen specifically to the phage is measuredby the change in signal generated by such a binding event, typically viaoptical, electrical or visual means. Antigen binding to the phage can beranked by concentration of the phage, off-rate of the antigen, and theability of the phage to functionally bind cells.

A phage preparation can be immobilized to a biosensor surface usingspecific antibody immobilization. An antibody to a phage coat protein isimmobilized to the biosensor surface. The antibody can be passivelyimmobilized to the biosensor surface or via a specific surface such asprotein A or a protein A plus anti-Fc surface. The surface can beblocked by a blocker. The binding of the phage specifically to thesurface is measured by the change in signal generated by such a bindingevent, typically via optical, electrical or visual means. The display onthe phage (virus) can be a peptide, small protein, and/or an antibodyfragment or non-existent. The binding of the cognate ligand is thensequentially measured by the change in signal generated by such abinding event, typically via optical, electrical or visual means. Theligand can be, e.g., a small molecule, carbohydrate, polymer, peptide,soluble protein, antigen mimic of a cellular receptor or a protein onthe surface of cells. The protein expressed on the surface of the cellcan be, e.g., a membrane-associated protein, a single ormulti-transmembrane protein, or a protein channel.

A phage preparation can be immobilized to a biosensor surface by anantigen bound to the biosensor surface. An antigen (such as a smallmolecule, carbohydrate, polymer, peptide, soluble protein, or antigenmimic of a cellular receptor) is immobilized on the biosensor surface ina passive or specific surface such as an antibody that does notinterfere with the desired binding epitope being screened. The antigensurface can be blocked. The binding of the phage preparationspecifically to the antigen is measured by the change in signalgenerated by such a binding event, typically via optical, electrical orvisual means. The display on the phage can be, e.g., a peptide, smallprotein, and/or an antibody fragment. Phage binding to the antigen canbe ranked by concentration of the antigen, and the off-rate of thephage.

Antibody fragments (and proteins) can be captured specifically to thesurface of the biosensor through biotin or proteinaceous tags (multiplehistidines, c-myc, or FLAG, MBP, GST_etc.) fused to their N- orC-terminal domains. A specific surface can be built on the biosensorbased on antibodies to the tags. Alternatively, antibody fragments canbe captured specifically through the constant region (CH1). A specificsurface can be built on the biosensor based on antibodies to this regionusing anti-lambda, anti-kappa, a mixture of anti-lambda and anti-kappa,and/or anti-F(ab′)₂ antibodies. The surface can be blocked. The bindingof the antibody fragment specifically to the antibody is measured by thechange in signal generated by such a binding event, typically viaoptical, electrical or visual means. The immobilization of the antibodyfragment can be from a pure or crude (whole cell extract, periplasmicextract, or spent media) sample. The binding of the cognate ligand canthen be sequentially measured by the change in signal generated by sucha binding event, typically via optical, electrical or visual means. Theligand can be, e.g., a small molecule, carbohydrate, polymer, peptide,soluble protein, antigen mimic of a cellular receptor or a protein onthe surface of cells. The protein expressed on the surface of the cellcan be, e.g., a membrane-associated protein, a single ormulti-transmembrane protein, or a protein channel.

While it is not necessary for specific binding substances or bindingpartners to comprise a detectable label, detectable labels can be usedto detect specific binding substances or binding partners on the surfaceof a biosensor. Where specific binding substances and binding partnersof the instant invention are free of detection labels, they can stillcomprise other types of labels and markers for enhancement of assaysensitivity, immobilization of specific binding partners to a biosensorsurface, enhancement of binding or hybridization of specific bindingsubstances to their binding partners, and for other purposes.

Molecules can be immobilized onto a biosensor so that they will not bewashed away by rinsing procedures, and so that binding to molecules in atest sample is unimpeded by the biosensor surface. Several differenttypes of surface chemistry strategies have been implemented for covalentattachment of molecules to, for example, glass for use in various typesof microarrays and biosensors. These same methods can be readily adaptedto a biosensor of the invention.

One or more types of molecules can be attached to a biosensor surface byphysical adsorption (i.e., without the use of chemical linkers) or bychemical binding (i.e., with the use of chemical linkers). Chemicalbinding can generate stronger attachment of molecules on a biosensorsurface and provide defined orientation and conformation of thesurface-bound molecules.

Other types of chemical binding include, for example, amine activation,aldehyde activation, and nickel activation. These surfaces can be usedto attach several different types of chemical linkers to a biosensorsurface. While an amine surface can be used to attach several types oflinker molecules, an aldehyde surface can be used to bind proteinsdirectly, without an additional linker. A nickel surface can be used tobind molecules that have an incorporated histidine (“his”) tag.Detection of “his-tagged” molecules with a nickel-activated surface iswell known in the art (Whitesides, Anal Chem. 68, 490, (1996)).

Immobilization of specific binding substances to plastic, epoxy, or highrefractive index material can be performed essentially as described forimmobilization to glass. However, an acid wash step can be eliminatedwhere such a treatment would damage the material to which the specificbinding substances are immobilized.

Antigens

One or more specific binding substances can be immobilized on abiosensor by for example, physical adsorption or by chemical binding. Aspecific binding substance can be, for example, an organic molecule,such as a nucleic acid, polypeptide, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus,phage, bacteria, polymer, peptide solutions, single- or double-strandedDNA solutions, RNA solutions, solutions containing compounds from acombinatorial chemical library, or biological sample; or an inorganicmolecule. A biological sample can be for example, blood, plasma, serum,gastrointestinal secretions, homogenates of tissues or tumors, synovialfluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinalfluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid,tears, or prostatic fluid. Preferably, an antibody, an antibodyfragment, an antigen, or a phage is immobilized on a biosensor. Suchspecific binding substances can be directly immobilized as describedherein, or can be indirectly immobilized via a specific surface. Forexample, an antibody or protein comprising a proteinaceous tag (e.g.histidines, c-myc, or FLAG, MBP, GST etc.) can be immobilized to abiosensor via an antibody that binds the tag. A binding partner is asubstance that specifically binds to a specific binding substance. Abinding partner can be any type of sample or molecule as described abovefor specific binding substances.

Methods

The methods of the invention can be used, for example, in: developmentof therapeutic and diagnostic antibodies by screening hybridomas, humanantibodies from mice and phage display technologies; screening mRNA T7phage display libraries; and a mammalian and bacterial cell free systemfor titration of bacterial as well as mammalian viruses. In thisdocument binding molecules will be discussed, in particular, antibodyfragments and/or full antibodies, eg. FIG. 1.

Applications for Viruses and Phage

The method of the invention can be used to titrate viruses. Typically,titration of viruses requires live mammalian and bacterial cells formammalian and bacterial viruses, respectively. The circles of death orslower growing cells are counted. Using methods of the inventionantibodies to the coat protein of the mammalian or bacterial virus areattached to the biosensor and dilutions of intact virus are detected viathe antibody and coat protein interaction. In addition, M13 phage can betitrated by direct immobilization of the phage using a GA3 high densitygluteraldehyde biosensor, see eg. FIG. 2. The GA3 biosensor was hydratedwith PBS for 30 minutes, then purified M13 in 20% glycerol were preparedusing 1:2 serial dilutions from 1.0e14 to 1.3e11 plaques per ml and wereadded to the sensor in 20% glycerol and PBS. The phage solutions wereincubated with the sensor for 2 hours. The unbound phage were washedaway and an endpoint reading was recorded for the amount of phageimmobilized via the free amines on the phage protein coat.

Methods of the invention can also be used in the investigation of thebiology (receptor binding, entry into cells, screening of neutralizingantibodies, etc.) surrounding mammalian viruses. For example, a virus isimmobilized on the biosensor and antibodies or proteins are screened fortheir ability to increase or decrease the fusion of the virus to themammalian cells.

The methods of the invention can also be used to detect toxins, viruses,and other bio-terrorism agents. For example, one can pan againsttargets, such as known toxins, virus, and other bio-terrorism agents orantigens derived from them with a phage display library. These librariescan display peptides, antibody fragments, protein scaffolds, or proteindomains. During the panning process, phage are enriched for the abilityto bind the target. During the screening process of the phage, leadcandidates are identified. At this point, methods of the invention canbe used to detect the toxins, virus, or other bio-terrorism agents byimmobilizing the phage to the biosensor. This would replace thecumbersome work of synthesizing the peptide or performing molecularcloning of the protein or antibody fragments that are displayed on thephage.

Methods of the invention can be used for screening (after a phagepanning or selection experiment) a large number of potential bindingdisplay phage for positive binding. Due to the ability to createspecific binding surfaces, phage can be specifically pulled out ofdefined and undefined solutions, such as bacterial extracts and spentmedia and immobilized on a biosensor.

Methods of the invention can also be used to rank antibody affinitiesearly at the phage level of screening, when using phage displaytechnologies to identify therapeutic or diagnostic antibodies. Forexample, a phage displaying an antibody fragment can be immobilized tothe biosensor directly or via an antibody to the coat protein. A signalfor the amount of phage immobilized per well is recorded. The antigenfor the antibody displayed on the phage is added and a signal isrecorded. A rank can be determined by dividing the signal/well forantigen by the signal/well for the phage. By this calculation theantigen signal is normalized for units of phage in each assay (well).The antibody fragment on the phage could be ranked for its monovalentaffinity without the use of molecular biology techniques to clone thegene for the antibody fragment. This technique would enable ranking ofantibody fragments with out the need for cloning the gene, expression ofthe antibody fragment, purification of the antibody fragment, thenranking of binding with the antigen.

Methods of the invention can be used to determine the off-rate of thedisplay on a phage for the corresponding antigen. For example, a phageis immobilized on a biosensor, washed, and antigen is bound. After eachwash sequence, the biosensor is read. If the antigen is released fromthe phage on the biosensor, then there is a loss in signal. Thebiosensor is monitored over multiple washes and the off-rate iscalculated as the loss of signal over time.

Binding partners can be identified using this technology, by bindingcellular components to the biosensor, then adding protein partners orphage containing DNA sequences corresponding to cellular components. Atest sample, such as cell lysates containing binding partners, can beapplied to a biosensor of the invention, followed by washing to removeunbound material. The binding partners that bind to a biosensor cansubsequently be eluted from the biosensor and identified by, forexample, mass spectrometry. A phage DNA display library can be appliedto a biosensor of the invention followed by washing to remove unboundmaterial. Individual phage particles bound to the biosensor can beisolated and the inserts in these phage particles can then be sequencedto determine the identities of the binding partners. Antibodies can beimmobilized in an array format onto a biosensor, which is then contactedwith a test sample of interest comprising binding partners, such asproteins. Only the proteins that specifically bind to the antibodiesimmobilized on the biosensor remain bound to the biosensor. Such anapproach is essentially a large-scale version of an enzyme-linkedimmunosorbent assay; however, the use of an enzyme or fluorescent labelis not required.

The methods of the invention provide several advantages overconventional phage display and phage panning protocols, including, forexample: (1) label free and direct binding of the display on the phageimmobilized on the biosensor to cells or other binding partners is notinfluenced by protein labels and amplification of secondary signals; (2)specific pull down of phage (i.e., immobilization of phage to abiosensor) from crude samples and the binding of cells or other bindingpartners without excessive washing detects functional binding ofmammalian cells faster; and (3) high throughput, thereby providing themost efficient and rapid phage screening methods.

In addition, conventional protocols using phage display to determinefunctional binding of antibodies to mammalian cells requires aconsiderable amount of time and effort in cloning the display on thephage to fragments and/or full IgGs in order to investigate thefunctional binding to cells. The methods of the invention simplify theprocess by eliminating the need to purify phage and are amenable to highthroughput screening as described herein. Thus, the methods of theinvention as described herein provide the benefit of allowing aresearcher to go from the phage panning experiment directly to testingthe functional activity of the display on mammalian cells (thefunctional antigen). In particular, since the methods of the inventionare high throughput, cellular binding can be investigated in the firstbinding experiment rather than later as in a conventional discoverypipeline. As the binding to cells is the crucial assay for identifying atherapeutic antibody, the methods of the invention enable a researcherto acquire critical information early, thereby accelerating therapeuticdiscoveries. In many cases the methods of the invention could savediscovery researchers up to three to six months of effort in identifyingtherapeutic antibodies.

For the above applications, and in particular proteomics applications,the ability to selectively bind material, such as binding partners froma test sample onto a biosensor of the invention, followed by the abilityto selectively remove bound material from a distinct location of thebiosensor for further analysis is advantageous. Biosensors of theinvention are also capable of detecting and quantifying the amount of abinding partner from a sample that is bound to a biosensor arraydistinct location by measuring the shift in reflected wavelength oflight. Additionally, the wavelength shift at one distinct biosensorlocation can be compared to positive and negative controls at otherdistinct biosensor locations to determine the amount of a bindingpartner that is bound to a biosensor array distinct location.

Applications for IgGs and Antibody Fragments

Methods of the invention can be used to capture antibodies and antibodyfragments, from complex media such as, e.g., periplasmic extracts,hybridoma supernatants, plasma, or sera. See, e.g., FIGS. 3 to 10. Thefollowing specific surface can be modified to capture full IgGs or F(ab)by substituting rabbit anti-mouse-Fc, rabbit anti-human Fc, rabbitanti-human F(ab′)2, or the rabbit anti-mouse-F(ab′)2 in the followingexample. The following procedure is for the capture of sF(ab) spikedinto PBS and periplasmic cultures described in FIG. 3. A specificcapture surface for sFab was created on a SRU BIND® TIO Biosensor, FIG.3(A). A hydrated TIO BIND® Biosensor was coated with 20 ug/ml Protein Ain PBS for 30 minutes, washed and an endpoint reading was recorded forthe amount of protein A deposited. The Biosensor was then blocked for 30minutes with 1% milk, washed and an endpoint reading was recorded forthe amount of milk deposited. Then 20 ug/ml of the specific capturereagent, rabbit anti-mouse F(ab′)2 specific IgG, was incubated for 30minutes, washed, and an endpoint reading was recorded for the amount ofrabbit anti-mouse F(ab′)2 specific IgG deposited. The remaining proteinA sites were blocked with 50 ug/ml rabbit IgG for 30 minutes. At thispoint a specific capture surface for mouse sF(ab) has been created. Inthis example, purified polyclonal mouse sF(ab), 0.33, 1.0 and 3.0 ug/ml,were spiked into PBS and into a Escherichia coli periplasmic prep, notexpressing sF(ab), FIGS. 3(A) and 4(B).

FIG. 4 shows scFv capture from periplasmic extract on a TIO BIND®Biosensor. A hydrated TIO BIND® Biosensor was coated with 20 ug/mlProtein A in PBS for 30 minutes, washed and an endpoint reading wasrecorded for the amount of protein A deposited. The Biosensor was thenblocked for 30 minutes with 1% milk, washed and an endpoint reading wasrecorded for the amount of milk deposited. Then 20 ug/ml of the specificcapture reagent, rabbit anti-mouse-Fc, was incubated for 30 minutes,washed, and an endpoint reading was recorded for the amount of rabbitanti-mouse-Fc specific IgG deposited. Two capture antibody surfaces werecreated, (1) 25 ug/ml anti-his and 25 ug/ml anti-cmyc and (2) 50 ug/mlanti-cmyc, incubated for 30 minutes, washed, and an endpoint reading wasrecorded for the amount of capture antibodies deposited. At this point ascFv capture surface has been created, FIG. 4(A). Purified scFv (5ug/ml) were spiked into PBS and periplasmic extracts of Escherichiacoli, not containing plasmids encoding scFv. The scFv were incubated for1 hour, washed, and an endpoint reading was recorded for the amount ofscFv deposited. The amount of scFv is tabulated in FIG. 4(B) andgraphically represented in FIGS. 4(C) and 4(D).

FIG. 5 shows scFv capture from periplasmic extract on a SA1 BIND®Biosensor. A hydrated SA1 BIND® Biosensor, streptavidin, was incubatedwith 50 ug/ml biotin anti-his for one hour, washed, and an endpointreading was recorded for the amount of biotin-anti-his deposited FIG.5(A). Eight micrograms per milliliter of scFv was spiked into PBS andperiplasmic extracts of Escherichia coli, not containing plasmidsencoding scFv. The scFv were incubated for 1 hour, washed, and anendpoint reading was recorded for the amount of scFv deposited. Theamount of scFv is tabulated in FIG. 5(B) and graphically represented inFIG. 5(C).

FIG. 6 shows scFv capture from periplasmic extract on a GA1 BIND®Biosensor. A hydrated GA1 BIND® Biosensor, gluteraldehyde, was incubatedwith 50 ug/ml anti-cmyc for one hour, washed, and an endpoint readingwas recorded for the amount of anti-myc deposited, FIG. 6(A). Twohundred micrograms per milliliter of neutravidin was incubated with theBIND® Biosensor to block any remaining gluteraldehyde reactive groupsfor 1 hour, washed, and an endpoint reading was recorded for the amountof neutravidin deposited. Five micrograms per milliliter of scFv wasspiked into PBS and periplasmic extracts of Escherichia coli, notcontaining plasmids encoding scFv. The scFv were incubated for 1 hour,washed, and an endpoint reading was recorded for the amount of scFvdeposited. The amount of scFv is tabulated in FIG. 6(B) and graphicallyrepresented in FIG. 6(C).

In FIG. 7, mouse IgGs created via hybridoma technologies are capturedfrom hybridoma supernatants then tested for their ability to bindsoluble antigen and antigen expressed on cells. A specific capturesurface for mouse IgG was created on a SRU BIND® TIO Biosensor, FIGS.7(A) and 7(B). A hydrated TIO BIND® Biosensor was coated with 20 ug/mlProtein A in PBS for 30 minutes, washed and an endpoint reading wasrecorded for the amount of protein A deposited. The biosensor was thenblocked for 30 minutes with 1% milk, washed and an endpoint reading wasrecorded for the amount of milk deposited. Then 20 ug/ml of the specificcapture reagent, rabbit anti-mouse Fc specific IgG, was incubated for 30minutes, washed and an endpoint reading was recorded for the amount ofrabbit anti-mouse Fc specific IgG deposited. The remaining protein Asites were blocked with 50 ug/ml rabbit IgG for 30 minutes. At thispoint a specific capture surface for mouse IgG has been created. Plate15 was used to test binding of antigen expressing transfected cell lineand the parental cell line. Plate 16 was used to test antigen binding.FIG. 7(C) shows the shift in nm measured for the capture of mouse IgGsfrom hybridoma supernatants on Plate 15 and Plate 16. FIG. 7(D) showsthe antigen and cell binding to the captured IgGs on the BIND®Biosensors. FIG. 7(E) shows the nm shifts for the antigen and cellbinding as well as the ratio of antigen signal divided by the IgG signaland the ratio of the cell binding signal divided by the IgG signal.These ratios can be used to rank the IgGs. Antibodies 22, 25, 26, 28,30, 31, 34, 36 and 37 can be classified as antigen binders (antigenbinding divided by IgG binding) with 37 having the highest affinity forthe antigen with a ratio of 0.13. Antibodies 22, 25, and 26 have similaraffinities and constitute the second class of binders with a ratio of0.10. The remaining antibodies (28, 30, 31, 34 and 36) have much loweraffinities with their ratio less than 0.04. The IgGs can also beclassified as a general cell binder in the case of antibody 30 with aratio for the parental cell line divided by IgG of 0.11 and the antigenpresenting cells divided by IgG of 0.16. The second cell binding classwould be specific for antigen expressed on the cells as in theantibodies 25 and 26 with ratios for the antigen presenting cellsdivided by IgG of 0.49 and 0.30, respectively, and the parental cellline divided by IgG of 0.00 and 0.01, respectively. The rest of theantibodies are non-cell binders with ratios for binding the parentalcell line divided by IgG and the antigen presenting cells divided by IgGless than 0.04.

FIG. 8 shows antibody capture from serum using an anti-Fc TIO BIND®Biosensor. A specific capture surface for mouse IgG was created on a SRUBIND® TIO Biosensor FIG. 8(A). A hydrated TIO BIND® Biosensor was coatedwith 20 ug/ml Protein A in PBS for 30 minutes, washed and an endpointreading was recorded for the amount of protein A deposited. TheBiosensor was then blocked for 30 minutes with 1% milk, washed and anendpoint reading was recorded for the amount of milk deposited. Then 20ug/ml of the specific capture reagent, rabbit anti-mouse Fc, wasincubated for 30 minutes, washed and an endpoint reading was recordedfor the amount of rabbit anti-mouse Fc deposited. The remaining proteinA sites were blocked with 50 ug/ml rabbit IgG for 30 minutes. At thispoint a specific capture surface for mouse IgG has been created. FIGS.8(B) and (C) shows the capture of mouse IgG (serial dilution from0.1-6.4 ug/ml) that were spiked into PBS and 5%, 10%, 20%, 40% and 100%serum.

FIG. 9 shows the Drug—anti-Drug assay in serum using a GA1 BindBiosensor. An IgG is the mimic for a therapeutic antibody drug and theanti-Drug is a F(ab′)2 spiked into PBS and mouse sera to mimic IgGsfound in human sera from clinical isolates using a GA1 BIND® Biosensor.A hydrated GA1 BIND® Biosensor was incubated with 20 ug/ml IgG for 1hour, washed and an endpoint reading was recorded for the amount IgGdeposited on the surface. The free aldehydes remaining on the surface ofthe GA1 BIND® Biosensor were bound up by incubating the sensor with 100ug/ml non-immune rabbit IgG for 2 hours. The IgG (Drug):rIgG surface wasalso blocked by START Block from Pierce for 1 hr. FIG. 9(A) shows the nmshifts for the production of a drug surface specific. A titration curveof anti-Drug from 0-7.1 ug/ml was incubated on the Biosensor by dilutingthe anti-drug in serial 1:3 dilutions into PBS or 11%, and 30% mouseserum. The binding of anti-Drug to the 20 ug/ml, FIG. 9(B) and to 0ug/ml Drug surface, FIG. 9(C) is represented in graphs. The same amountof anti-drug is captured in 11% serum compared to PBS. Less anti-Drug iscaptured in 30% serum, but the anti-Drug is detectable.

FIG. 10 shows the development of a Drug—anti-Drug assay in serum using aTIO BIND® Biosensto. An IgG is the mimic for a therapeutic antibody drugand the anti-Drug is a F(ab′)2 spiked into PBS and mouse sera to mimicIgGs found in human sera from clinical isolates. FIG. 10(A) shows theshifts measured during the creation of a specific surface for thecapture of mouse IgGs. A hydrated TIO BIND® Biosensor is coated with 20ug/ml of protein A. One percent milk is used to block any remainingbinding sites on the TIO not filled by protein A. After the milkblocking step the surface is incubated with 20 ug/ml rabbitanti-human-Fc. Rabbit IgG at 50 ug/ml is used to block any protein Abinding sites not filled by the rabbit anti-mouse-Fc. During this stageof the experiment each reagent is incubated with the surface for thirtyminutes, the surface is washed and an endpoint reading was recorded forthe amount of each reagent deposited on the surface. Fifty microgramsper milliliter of Drug (IgG) was incubated with the specific surface for1 hour, washed and an endpoint reading was recorded for the amount ofeach Drug deposited on the surface. A titration curve of anti-Drug from0-7.1 ug/ml was incubated on the Biosensor by diluting the anti-drug inserial 1:3 dilutions into PBS or 11%, and 30% mouse serum. The bindingof anti-Drug to the 50 ug/ml and 0 ug/ml Drug surface is shown in FIGS.10(B) and 11(C), respectively. Less anti-Drug is captured in 11% and 30%serum, but the anti-Drug is detectable.

Determination of Epitope Classes within an Antibody Screen

Epitope classes within an antibody screen (antibody or antibodyfragment) or a phage screen can be determined using methods of theinvention. In the example, the binding molecules to be classified can beantibodies or display phage or a combination of both. The first bindingmolecule is passively or specifically immobilized to a biosensor.Preferably, the biosensor comprises the bottom surface of a microtiterplate. The surface can be blocked by blockers. An antigen (such as acarbohydrate, polymer, peptide, soluble protein, or antigen mimic of acellular receptor) is captured specifically by the display phage,antibody, or antibody fragment surface. Individual phage clones,antibodies, or antibody fragments to be classified are added to thewells. Where the display phage, antibody, or antibody fragmentimmobilized in the wells bind within the same epitope class as thephage, antibody or antibody fragment to be classified, there would notbe a measurable signal as the binding sites are filled via theimmobilized phage, antibody or antibody fragment. Where the displayphage, antibody, or antibody fragment immobilized in the wells recognizedifferent epitope binding sites than the phage, antibody or antibodyfragment to be classified then a measurable signal would be recorded.

FIG. 11 shows an endpoint analysis of monoclonal antibody binding targetpeptides for epitope mapping using a calorimetric resonant reflectancebiosensor. FIG. 11(A) shows the shifts measured during the creation of aspecific surface for the capture of mouse IgGs. A hydrated TIO BIND®Biosensor is coated with 20 ug/ml of protein A. The protein A acts as acapture surface for the rabbit anti-mouse-Fc that will specificallycaptures mouse IgGs. One percent milk is used to block any remainingbinding sites on the TIO not filled by protein A. After the milkblocking step the surface is incubated with 20 ug/ml rabbitanti-mouse-Fc. Rabbit IgG at 50 ug/ml is used to block any protein Abinding sites not filled by the rabbit anti-mouse-Fc. During this stageof the experiment each reagent is incubated with the surface for thirtyminutes, the surface is washed and an endpoint reading was recorded forthe amount of each reagent deposited on the surface. In FIG. 11(B), thecapture of the four mouse IgGs by the protein A:milk:rabbitanti-mouse-Fc:rabbit IgG surface is recorded. Ten micrograms permilliliter of IgGs were incubated with the specific surface for 30minutes. To ensure that the second layer of mouse IgGs bind through theantigen and not the rabbit anti-mouse-Fc, non-immune mouse IgG fromPierce is added at 50 ug/ml to fill any unoccupied rabbit anti-mouse-Fcbinding sites, FIG. 11(A). FIG. 11(C) records the shift measured whenantigen is added to captured mouse IgG. Ab-4 does not bind antigen andthe 4 IgGs can be classified as antigen binders (Ab-1, Ab-2 and Ab-3)and antigen non-binders (Ab-4). The antigen binding antibodies can alsobe ranked by dividing the antigen shift by the antibody shift measuredby the BIND® Biosensor. Ab-2 has the highest affinity for antigen with aratio of 0.56. Ab-3 has a slightly higher affinity for the antigen thanAb-1 with ratios of antigen binding per antibody shift of 0.38 and 0.35,respectively. FIG. 11(D) shows the grid created by the addition of thesame mouse IgGs in layer 1 as layer 3. If two IgGs in the same well bindthe same area of the antigen, then no signal will be measured. This isexemplified by the addition of the same antibody into the same welltwice. Self competition results in no signal as seen on the diagonal:Ab-1 vs. Ab-1 results in 0.016 nm; Ab-2 vs. Ab-2 results in 0.063 nm;and Ab-3 vs. Ab-3 results in 0.050 nm. Ab-1 and Ab-3 are in the samebinding class and cannot be used as a sandwich pair as Ab-1 vs Ab-3results in 0.033 nm shift and reverse orientation of Ab-3 vs. Ab-1results in 0.024 nm shift. If the two IgGs in a well are both able tobind the antigen simultaneously, then they belong to different bindingclasses and can be used as a sandwich pair. Ab-2 is in a second bindingclass and can be used as a sandwich partner for both Ab-1 and Ab-3: Ab-1vs. Ab-2 equals 0.138; Ab-2 vs. Ab-1 equals 0.150; Ab-3 vs. Ab-2 equals0.203; and Ab-2 vs. Ab-3 equals 0.212. This technique will work wherethe first and/or the second antibody immobilized are a displayed onphage or a soluble antibody (purified or crude sample). Combinationsinclude phage:phage, phage:antibody fragment, antibody fragment:phage,antibody fragment:antibody fragment, full antibody:phage, phage:fullantibody, full antibody:full antibody, antibody fragment:full antibody,and antibody:antibody fragment. This can also be applied to hybridomaand phage display screens as well as human antibodies made in otherhosts, such as mice.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The methods and compositions described herein aspresently representative of preferred embodiments are exemplary and arenot intended as limitations on the scope of the invention. Changestherein and other uses will be evident to those skilled in the art, andare encompassed within the spirit of the invention. The inventionillustratively described herein suitably can be practiced in the absenceof any element or elements, limitation or limitations that are notspecifically disclosed herein. Thus, for example, in each instanceherein any of the terms “comprising”, “consisting essentially of”, and“consisting of” can be replaced with either of the other two terms,while retaining their ordinary meanings. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by embodiments and optional features,modification and variation of the concepts herein disclosed areconsidered to be within the scope of this invention as defined by thedescription and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

1. A method for detecting if different epitope classes of antibodies inan antibody population are present as compared to display phage,antibodies, or antibody fragments immobilized on a biosensor comprising:(a) immobilizing display phage, antibodies, or antibody fragments to abiosensor; (b) contacting the biosensor with a binding partner thatspecifically binds to the display phage, antibodies, or antibodyfragments immobilized to the biosensor, under conditions suitable forbinding of the binding partner to the display phage, antibodies, orantibody fragments; (c) contacting the antibody population with thebiosensor; wherein, a detectable signal generated by binding of theantibody population to the binding partner indicates that differentepitope classes are present in the antibody population than in theimmobilized display phage, antibodies, or antibody fragments.
 2. Themethod of claim 1, wherein the antibody population comprises phageclones.
 3. The method of claim 1, wherein the antibody populationcomprises antibody fragments, full antibodies, phage displaying a fullantibody, or phage displaying an antibody fragment.
 4. The method ofclaim 1, wherein the antibody population comprises antibodies from ahybridoma.
 5. The method of claim 1, wherein the antibody populationcomprises antibodies from a phage display screen.
 6. The method of claim1, wherein the display phage is a crude phage preparation, anunconcentrated phage preparation, a concentrated phage preparation or anon-homologous phage preparation.
 7. The method of claim 1, wherein thebinding partner is a small molecule, a carbohydrate, a polymer, apeptide, a soluble protein, a cellular receptor, an antigen mimic of acellular receptor, a mammalian cell, or a mammalian cell surfaceprotein.
 8. The method of claim 7, wherein the mammalian cell surfaceprotein is a membrane-associated protein, a single transmembraneprotein, a multi-transmembrane protein, or a protein channel.
 9. Themethod of claim 1, wherein the biosensor is a colorimetric resonantreflectance biosensor or an evanescent wave-based biosensor.
 10. Themethod of claim 1, wherein the antibody population, binding partner andimmobilized display phage, immobilized antibody, or immobilized antibodyfragment do not comprise a detectable label.
 11. The method of claim 1,wherein the display phage, antibodies, or antibody fragments areimmobilized in an array format on the biosensor.
 12. The method of claim1, wherein the biosensor is incorporated into a bottom surface of amicrotiter plate comprising wells, and wherein the display phage,antibodies, or antibody fragments are immobilized to the biosensorwithin each well.
 13. The method of claim 1, where the method furthercomprises determining the quantity of binding partner bound to the phageimmobilized on the biosensor.
 14. The method of claim 1, wherein thebiosensor is coated with streptavidin, amines, aldehydes orgluteraldehydes prior to the immobilization of the display phage,antibodies, or antibody fragments to the biosensor.